PROJECT SUMMARY/ABSTRACT Cigarette smoking is a known risk factor for lung cancer, yet is also common among lung cancer patients. Electronic nicotine delivery systems (?ENDS?), i.e., ?E-cigarettes?, are rapidly growing in popularity as a safer alternative to cigarettes to aid in tobacco cessation efforts, however, ?safer? does not necessarily mean ?safe?. There is a knowledge gap in our understanding of the molecular structures that comprise ENDS aerosols and the biological consequences of ENDS aerosols. This information is critically needed to develop an early understanding of the potential adverse health effects of ENDS in humans. To address this knowledge gap, the following two Specific Aims are proposed. Specific Aim 1: Investigation of the formation mechanism of ENDS aerosols at different temperatures, which will be enabled by using our recently developed in situ (a few kHz) magic angle spinning (MAS) technique that is capable of generating high resolution NMR spectra on samples containing a mixture of gases, liquids, and solids at significantly elevated temperature (from 0 to > 250?C) and pressure (below 1 bar to >100 bars). This unique technique is ideal to determine how sensitive different E-liquid components are to aerosolization as a function of the temperature, including in particular the highly toxic chemicals such as aldehydes that can be generated by pyrolysis of ENDS solvents at high temperatures, and to determine the molecular structure of aerosols produced at different temperatures. Specific Aim 2: Application of non-destructive slow-MAS NMR metabolomics platform to define the dynamic response of lung organotypic cultures to ENDS aerosols. Slow-MAS NMR dramatically increases spectral resolution on intact biological tissues and cells, allowing detection of more metabolite features than can be resolved by conventional NMR instruments. The non-destructive capability of slow-MAS NMR (40-100Hz) is also uniquely suited for extended live cell/tissue measurements which is far superior to destructive approaches examining single time points. The feature of non-destructive detection is particularly advantageous as some metabolites only exist in live biological systems. We have developed a lung organotypic culture platform that enables us to investigate single cell populations (e.g. normal vs cancer cells), as well as mixed cell populations (e.g. normal/cancer cell co-cultures). We will use this system to define the baseline metabolome of normal human lung epithelial cells, lung cancer cells and their mixture as cocultures, as well as dynamic changes in these experimental groups induced by ENDS aerosols generated at different temperatures. Indices of toxic potential (cell viability, stress-responsive gene expression) will be defined under identical conditions. The goal is to determine if the production of ENDS at different temperatures alters the constituents and/or molecular structure of ENDS aerosols and their toxic potential on human cell systems by discovering and utilizing metabolic signatures.